Data Sheet
Contents
1. Cat CY R2056 Glucose AGE OVA Cat CY R2057 Glyceraldehyde AGE BSA Cat CY R2058 Glyceraldehyde AGE OVA Cat CY R2059 Glycolaldehyde AGE BSA Cat CY R2060 Glycolaldehyde AGE OVA Cat CY R2061 Methylglyoxal AGE BSA Cat CY R2062 Methylglyoxal AGE OVA Cat CY R2063 Glyoxal AGE BSA Cat CY R2064 Glyoxal AGE OVA Cat CY R2065 CML HSA N Carboxymethyl lysine HSA Cat CY R2066 CEL HSA N Carboxyethyl lysine HSA Cat CY R2067 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 8066 13 Version 1402052. reagents are handled e Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary e Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8066 4 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifuge the samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma by use of EDTA Nay as the anticoagulant If possible collect the plasma into a mixture
3. significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Cat CY 8066 8 Version 140205 p i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product CircuLex CML N Carboxymethyl lysine ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted CML HSA Standard must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of CML HSA giving absorbance lower than mean absorbance plus three standard deviations of the absorbance of Blank Blank 3 SD Bl
4. temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 6 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 7 Add 100 pL of HRP conjugated Detection Antibody into each well 99 Incubate the wells at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 9 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 10 Add 100 uL of Substrate Reagent Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 11 Incubate the wells at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation time may be extended up to 30 minutes if the reaction temperature is below than 20 C 12 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 13 Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding
5. 66 Sapmle 1 60 74 72 44 58 04 72 4 58 0 63 7 7 7 12 0 10 Sapmle 2 136 80 131 41 124 47 136 8 124 5 130 9 6 2 4 7 Sapmle 3 190 90 247 41 255 43 255 4 190 9 231 2 35 2 15 2 Version 140205 CML N Carboxymethyl lysine ELISA Kit C ire uL ex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Concentrations of CML N Carboxymethyl lysine in patient afflicted lifestyle related disease and healthy volunteers serum 25 T20 E ob amp lt e Ww e S15 gt Q z 8 e w E 8 S 10 8 5 8 S 4 i amp 3 0 p 8 2 5f fom Q 0 Healthy Patient afflicted volunteers lifes tyle related serum disease serum n 39 n 39 Cat CY 8066 11 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures References Ikeda K Higashi T Sano H Jinnouchi Y Yoshida M Araki T Ueda S Horiuchi S Biochemistry 35 8075 8083 1996 2 Reddy S Bichler J Wells Knecht KJ Thorpe SR Baynes JW Biochemistry 34 10872 10878 1995 3 Makino H Shikata K Hironaka K Kushiro M Yamasaki Y Sugimoto H Ota Z Araki N Horiuchi S Kidney Int 48 517 526 1995 4 Suzuki D Yagame M Jinde K Naka R Yano N Endoh M Kaneshige H Nomoto Y Sakai H J Diabetes Complications 10 314 319 1996 5 Imai N Nish
6. ank is better than 0 063 ng mL of sample Typical Standard Curve Standard curve 2 0 1 5 1 0 0 5 0 0 0 0 0 1 1 0 10 0 CML HSA pg ml Cat CY 8066 9 Version 140205 CML N Carboxymethyl lysine ELISA Kit User s Manual CircuLex For Research Use Only Not for use in diagnostic procedures 2 Specificity The antibodies in the Circulex CML N Carboxymethyl lysine ELISA Kit are highly specific of CML adduct with no detectable cross reactivity to non CML protein that may be present in human serum plasma 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested seven times on one plate to assess intra assay precision e Intra assay Within Run n 7 CV 5 2 7 4 Yo Corresponding concentration of CML HSA ug mL NYDN BWN max min mean SD CV Sapmle 1 79 61 70 61 76 10 68 68 68 35 79 97 80 83 80 8 68 4 74 9 5 5 74 Inter assay Precision Precision between assays Three samples of known concentration were tested in three separate assays to assess inter assay precision e Inter assay Run to Run n 3 CV 4 7 15 2 Yo Sapmle 2 123 28 133 71 123 78 121 10 133 72 133 03 138 55 138 5 121 1 129 6 6 7 5 2 Sapmle 3 259 82 237 87 244 76 260 97 227 70 231 16 269 80 269 8 227 7 247 4 16 3 6 6 Corresponding concentration of CML HSA ug mL 1 2 3 max min mean SD CV Cat CY 80
7. ception of 10X Wash Buffer Primary Antibody and CML HSA Standard j Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water ddH O Mix well Store at 4 C for two weeks or 20 C for long term storage N Reconstitute Primary Antibody with 1 25 mL of ddH O and mix well Prepare an appropriate aliquot of Primary Antibody Working Solution by 30 fold diluting Primary Antibody with Sample Dilution Buffer e g 3 uL Primary Antibody 87 uL Sample Dilution Buffer 60uL per one well is required for assay Note Unused portions of Primary Antibody solution should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles 3 Reconstitute CML HSA Standard with 0 77 mL of ddH O The concentration of the CML HSA in vial should be 21 pg mL which is referred as a Master Standard of CML HSA Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 7 0 w ml standard Std 1 serves as the highest standard The Standard Dilution Buffer serves as the zero standard Blank Volume of Standard Standard Dilution Buffer Concentration Std 1 200 uL of Master Standard 400 uL 7 0 pg mL Std 2 300 pL of Std 1 7 0 pg mL 300 pL 3 5 ug mL Std 3 300 uL of Std 2 3 5 ug mL 300 pL 1 75 pg mL Std 4 300 uL of Std 3 1 75 ug mL 300
8. i S Suzuki Y Karasawa R Ueno M Shimada H Kawashima S Nakamaru T Miyakawa Y Araki N Horiuchi S Gejyo F Arakawa M Nephron 76 153 160 1997 6 Murata T Nagai R Ishibashi T Inomuta H Ikeda K Horiuchi S Diabetologia 40 764 769 1997 7 Kume S Takeya M Mori T Araki N Suzuki H Horiuchi S Kodama T Miyauchi Y Takahashi K Am J Pathol 147 654 667 1995 oo Sakata N Imanaga Y Meng J Tachikawa Y Takebayashi S Nagai R Horiuchi S Itabe H Takano T Atherosclerosis 141 61 75 1998 9 Sakata N Imanaga Y Meng J Tachikawa Y Takebayashi S Nagai R Horiuchi S Atherosclerosis 142 67 77 1999 10 Kislinger T Fu C Huber B Qu W Taguichi A Du Yan S Hofmann M Yan SF Pischetsrieder M Stern D Schmidt AM J Biol Chem 274 31740 31749 1999 Cat CY 8066 12 Version 140205 i i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex CML N Carboxymethyl lysine ELISA Kit Cat CY 8066 CircuLex Anti CML mouse autoantibody ELISA Kit Cat CY 8067 CircuLex Anti CML human autoantibody ELISA Kit Cat CY 8068 CircuLex Anti CML rat autoantibody ELISA Kit Cat CY 8069 CML BSA N Carboxymethyl lysine BSA Cat CY R2052 CML OVA N Carboxymethyl lysine OVA Cat CY R2053 CEL BSA N Carboxyethyl lysine BSA Cat CY R2054 CEL OVA N Carboxyethyl lysine OVA Cat CY R2055 Glucose AGE BSA
9. i i CML N Carboxymethyl lysine ELISA Kit C 1rcuL eX User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring CML N Carboxymethyl lysine CircuLex CML N Carboxymethy lysine ELISA Kit Cat CY 8066 Intended Ui 1 SONAR NI 1 IntrodUctionN wwwwwwwmmmenwwwmemwwwza 2 Principle of the Assay wswwwwwwwwwe 2 Materials Provided lt lt lt lt lt ve 3 Materials Reguired but not Provided 3 Precautions and Recommendations 4 Sample Collection and Storage 5 Detailed Protocol lt lt wes 6 8 CAI NE KAA UU AAA 8 Measurement Range w wwwwe 8 Troubleshooting lt w wwwwsmwmmemwmmswa 8 Reagent Stability lt wwwwwwwwemmwewe 9 Assay CharacteristicS lt 9 10 Example of Test ResultS lt lt lt lt 11 Ri ii 12 Related ProductS lt lt w wewe 13 Intended Use The CycLex Research Product CircuLex CML N Carbozymethyl lysine ELISA Kit is used for the quantitative measurement of CML adducts in mammalian serum plasma tissue extract and other biological media except rodent specimen This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon
10. ible but not preferable e Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm which will give a somewhat higher reading Software package facilitating data generation and analysis optional e 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels Cat CY 8066 3 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware e Use deionized water of the highest quality Do not mix reagents from different kits e The buffers and reagents used in this kit contain NaN as preservatives Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in areas where samples or
11. icroplate kit Antigen coated microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with CML BSA 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Sample Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample dilution and Primary Antibody dilution Ready to use Standard Dilution Buffer One bottle containing 10 mL of 1X buffer use for standard dilution Ready to use CML HSA Standard One vial containing 16 2 ug of lyophilized CML HSA Primary Antibody One vial containing lyophilized anti CML adduct monoclonal antibody MK 5A10 HRP conjugated Detection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti mouse IgG polyclonal antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle supplied ready to use containing 20 mL of 1 N H SO4 Materials Required but not Provided e Sample preparation microplate 96 wells microplate for mixing Primary Antibody and diluted samples e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer e Microplate washer optional Manual washing is poss
12. oclonal antibody present is bound by the immobilized CML BSA After washing away any unbound substances an HRP conjugated polyclonal antibody specific for mouse IgG is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H O gt tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is inversely proportional to the concentration of CML adduct A standard curve is constructed by plotting absorbance values versus CML adduct concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Summary of Procedure Mix 50 uL of diluted samples and 50 uL of anti CML adduct monoclonal antibody Add the mixture to each well 4 Incubate for 1 hour at room temp Wash the wells y Add 100 uL of HRP conjugated anti mouse IgG polyclonal antibody 4 Incubate for 1hour at room temp Wash the wells t Add 100 uL of Substrate Reagent 4 Add 100 uL of Stop Solution y Measure absorbance at 450 nm Cat CY 8066 2 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are supplied and are sufficient for the one 96 well m
13. of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Other biological samples Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles Cat CY 8066 5 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product Circulex CML N Carboxymethyl lysine ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the CML HSA Standard within the kit should be included in each assay as a calibrator Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay reagents are supplied ready to use with the ex
14. pL 0 875 pg mL Std 5 300 uL of Std 4 0 875 ug mL 300 pL 0 438 pg mL Std 6 300 uL of Std 5 0 438 ug mL 300 pL 0 219 ng mL Std 7 300 uL of Std 6 0 219 wg mL 300 pL 0 109 pg mL Blank 300 uL 0 pg mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Standard Dilution Buffer before dispensing Unused portions of Standards should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation Cat CY 8066 6 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C 1rcuL eX User s Manual For Research Use Only Not for use in diagnostic procedures Serum and plasma samples require 4 fold dilution with Sample Dilution Buffer Assay Procedure en Remove the appropriate number of microtiter wells of Antigen coated microplate from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Sample Dilution Buffer in appropriate wells of a sample preparation microplate U Pipette 60 uL of Standard Solutions Std1 Std7 Blank and the diluted samples in duplicates into the appropriate wells of a sample preparation microplate Pipette 60 uL of Primary Antibody Working Solution into each well Mix well 4 Transfer 100 uL of the mixtures prepared above to each well of Antigen coated microplate Nn Incubate the wells at room
15. rd curve At the point of intersection extend a vertical line to the x axis and read the corresponding CML HSA concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 5 parameter logistic equation The results of unknown samples can be calculated with any computer program having a 5 parameter logistic function It is important to make an appropriate mathematical adjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 109 ug mL to 7 0 ng mL Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the CML HSA concentration Troubleshooting The Standard Solutions should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures
16. receipt store all components at 4 C e Don t expose reagents to excessive light Cat CY 8066 1 Version 140205 i CML N Carboxymethyl lysine ELISA Kit C eu CX User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Reducing sugars react with protein amino groups to form a diverse group of protein bound moieties with fluorescent and cross linking properties These compounds called advanced glycosylation end products AGEs have been implicated in the structural and functional alterations of proteins that occur during aging and long term diabetes Although several AGE structures have been reported 1 2 it was demonstrated that N Carboxymethyl lysine CML is a major antigenic AGE structure CML concentration is also increased in patients who have diabetes with complications including nephropathy 3 5 retinopathy 6 and atherosclerosis 7 9 CML is also recognized by receptor for AGE RAGE and CML RAGE interaction activates cell signaling pathways such as NF B and enhances the expression of vascular cell adhesion molecule 1 in human umbilical vein endothelial cells 10 Principle of the Assay The CircuLex CML N Carboxymethyl lysine ELISA Kit employs the quantitative competitive enzyme immunoassay technique CML BSA has been pre coated onto a microplate Standards or samples and anti CML adduct monoclonal antibody MK 5A10 are pipetted into the wells Any free anti CML adduct mon
17. the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 2 0 units for the blank zero concentration The plate should be monitored at 1 minute intervals for approximately 15 minutes Cat CY 8066 7 Version 140205 CML N Carboxymethyl lysine ELISA Kit C ire uL ex User s Manual For Research Use Only Not for use in diagnostic procedures Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the blank zero concentrations perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine CML adduct concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Calculations Average the duplicate readings for each Standard Solution control and sample Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and regression analysis may be applied to the log transformation To determine the CML adduct concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standa
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